The carbohydrate antigens Globo H, stage-specific embryonic antigen-3 (SSEA-3), and stage-specific embryonic antigen-4 (SSEA-4) are closely related to one another in either structure or in function. Globo H, SSEA-3 and SSEA-4 are globoseries glycosphingolipids, with SSEA-3 being the non-fucosylated pentasaccharide precursor structure of Globo H, SSEA-4 is sialylated SSEA-3 with sialic acid α2-3 links to the non-reducing end of galactose of SSEA-3.
Stage specific embryonic antigen-3 (SSEA-3) was first identified and defined by the reactivity of an IgM monoclonal antibody generated in a rat immunized with 4-to 8-cell stage mouse embryos. This monoclonal antibody reacted with all mouse preirnplantation embryos from oocytes up to the early blastocyst stage where its expression became more restricted, in the primitive endoderm after implantation. The SSEA-3 antigenic determinant was determined to be a carbohydrate present on glycolipids and glycoproteins; it was also found on human teratocarcinoma cells and human erythrocytes. In a panel of structures isolated from the 2102Ep human teratocarcinoma cell line, the SSEA-3 antibody had the highest affinity for Galβ(1-3)GalNAcβ(1-3)Galα(1-4)Galβ(1-94)Glcβ(1)Cer. This structure is also known as Gb5, galactosyl-globoside, or globopentaosylceramide.
Synthesis of SSEA-3 occurs when β1,3-galactosyltransferase V (β3GalT-V) transfers galactose to the GalNAc of globoside to form Gb5 or galactosyl-globoside. In more recent studies, attempts were made to determine if SSEA-3 could be used as a marker to identify stem cells in umbilical cord blood. It was determined that SSEA-3 was not expressed in hematopoietic or mesenchymal stem cells and therefore was not a good marker of multipotent cells. Schrump et al. immortalized lymph node lymphocytes from primary lung cancer patients, generated hybridomas, and selected for antibody secreting clones. Monoclonal antibodies were then generated from two of these clones—J309 and D579, which recognized the SSEA-3 antigenic determinant. The antibodies recognized SSEA-3 on several tumor cell lines including Jung and breast cancer cell lines, and a teratocarcinoma cell line; in an immune adherence assay, rodent monoclonal SSEA-3 antibody, also referred to as MC631, reacted against the same cell lines as the J309 and D579 antibodies. SSEA-3 has also been found on testicular germ cell tumors, as well as in breast cancer and in BCSCs (breast cancer stem cells).
Chang et al. looked at SSEA-3 expression on normal tissues using a tissue microarray because its location outside of cancer and development was largely unknown. The group found SSEA-3 to be expressed on normal epithelium of colon, esophagus, small intestine, kidney, prostate, rectum, skin, testis, thymus, and uterine cervix. Expression was located only on the apical surfaces of epithelial cells or in the cytoplasm, which are considered immune system restricted or inaccessible sites.1 In an experiment using a KLH conjugated Globo H monovalent vaccine in mice, an antibody response was made to only the Globo H antigen. When α-GalCer was added as an adjuvant, the amount of overall antibody production increased and the mice made polyclonal antibodies to both the Globo H, the SSEA-3 and the SSEA-4 antigen structures, which vaccination was unable to generate in the absence of the adjuvant.1 This result showed that SSEA-3, Globo H and SSEA-4 could make promising targets for cancer vaccines and could be targeted simultaneously.
However, most tumor associated carbohydrate antigens have poor immunogenicity and many approaches have been developed to increase the immune response of carbohydrate-based vaccines, including conjugation with a carrier protein administration with an immunologic adjuvant using unnatural glycosidic linkage, clustered antigens, unimolecular polyvalent vaccine or hetero-glycan multivalent vaccine. Using these strategies, a few carbohydrate-based vaccines that could elicit significant immune responses to target glycan structures were designed for cancer therapy and entered clinical trials. Among them, the clinical trials of Theratope and GMK with adjuvant QS-21 failed to produce statistically significant difference between time-to-disease and overall survival rate. Probably these two vaccines could not elicit robust T cell-dependent immune response in patients. Specifically, Theratope and GMK induced a higher level of IgM in patients but could not induce a strong immune IgG response, which is a major problem in carbohydrate-based vaccine development.
Previous studies showed that modification of carbohydrate antigen structures (MCAS) could effectively elicit a higher level of immune response. For example, in the modification study of the capsular polysaccharide of group B meningococci, the N-acetyl groups of α-(2,8)-linked polysialic acid (PSA) was replaced with the N-propinoyl group and such a modification elicited a high antibody response to recognize not only the N-propinoyl PSA, but also the nature N-acetyl PSA. Similar approaches were applied to STn and GM3 antigens to produce high antibody titers against modified and nature forms. The results indicated that N-phenylacetyl, N-fluoroacetyl or N-difluoroacetyl modifications on glycan antigens could improve the immunogenicity. Moreover, the Schultz group reported that incorporation of a p-nitrophenylalanine into the tumor necrosis factor-α(TNF-α) could break immune tolerance and induce more antibody response to TNF-α. Using glycans as antigens, although some progress has been achieved, most cases are the N-modification of disaccharide (STn). trisaccharide (GM3) and polysialic acid (PSA) and some are based on fluorinated MUC1glycopeptide antigens.
The discovery of cancer stem cells (CSCs), which are responsible for self-renewal and tumor-growth in heterogeneous cancer tissues, has stimulated interests in developing new cancer therapies and early diagnosis. The markers currently used for isolation of CSCs, however, are often not selective enough to enrich CSCs for the study of this special cell population.